CN114062532A - Rheumatoid arthritis blood diagnosis kit and application thereof - Google Patents

Abstract

The invention relates to a rheumatoid arthritis blood diagnosis kit and application thereof, wherein the diagnosis kit comprises a reagent for detecting substances in a marker pool, and the marker pool comprises the following substances: dehydroepiandrosterone glycosulfate, glycine-L-proline, isovaleric acid, lactic acid, caprylic acid, and glycochenodeoxycholic acid. The invention provides a marker pool of a new metabolome in blood and a corresponding diagnostic kit, the kit can be used for diagnosing early rheumatoid arthritis by detecting metabolites in blood, has high accuracy, can discover the rheumatoid arthritis as early as possible, can classify the rheumatoid arthritis, can distinguish the rheumatoid arthritis from osteoarthritis, and can intervene and treat diseases as early as possible.

Description

Rheumatoid arthritis blood diagnosis kit and application thereof

Technical Field

The invention belongs to the technical field of life science or inspection methods, and relates to a rheumatoid arthritis blood diagnosis kit and application thereof.

Background

The information in this background section is only for enhancement of some understanding of the general background of the invention and is not necessarily to be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Rheumatoid Arthritis (RA) is a chronic autoimmune disease with joint synovial malignant lesions as a major feature, affecting about 1% of people worldwide. The highly destructive nature of the disease can ultimately lead to progressive sexual dysfunction, systemic complications and premature death. However, the pathogenesis of RA is not well understood, and there are no different stage diagnosis tools that prevent effective management. Currently, in clinical tests for RA, it is common to determine whether a patient suffers from rheumatoid arthritis by detecting combinations of clinical symptoms and rheumatoid factors (RF-IgM), anti-Cyclic Citrulline (CCP) antibodies, rheumatoid factors IgG and IgA, etc. in the peripheral blood of the patient. Blood is in contact with a variety of tissues and organs through circulation, providing for systemic expression of endogenous processes. Inevitably, analyzing possible RA markers in blood becomes a difficult task. Also, rheumatoid arthritis is similar to the clinical symptoms of Osteoarthritis (OA) and is not easily distinguished. Therefore, it is important to understand the complex molecular processes that play a role in RA pathogenesis, as well as to find new specific markers useful for its different stage diagnosis and monitoring.

Omics technology is widely used as a useful tool for molecular analysis, identification of biorarker, and examination of pathophysiological processes of various diseases. However, since biochemical regulation of disease is often a complex regulation at multiple levels, biological interpretation of monoomics research data is difficult.

Disclosure of Invention

Against the above background, the inventors thought that under disease conditions, changes in blood components may reflect pathophysiological events within large joint structures, blood containing metabolites, and thus the inventors of the present invention used blood as a source of candidate markers for joint diseases.

In the invention, the development of RA is known by comprehensively analyzing the change of blood environment by using metabonomics. The classification criteria in 2010, according to the American College of Rheumatology (ACR) and european antirheumatic union (EULAR), were classified into RAI, RAII and RAII, and the necessary identified pool of RA new markers was analyzed in combination with the RA clinical phenotype. The results of the study contribute to a comprehensive understanding of the pathogenesis of RA.

In view of the above, the present inventors have invented a new blood marker pool as an early diagnosis kit for rheumatoid arthritis and related applications.

Specifically, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a use of the following substances as a marker pool in preparing or screening a rheumatoid arthritis diagnostic reagent or an early diagnosis kit, wherein the marker pool comprises the following substances: dehydroepiandrosterone glycosulfate (Dehydroepiandrosterone sulfate), glycine-L-proline (glycoproline), Isovaleric acid (Isovaleric acid), Lactic acid (Lactic acid), Caprylic acid (Caprylic acid), and glycochenodeoxycholic acid (Chenodeoxycholic acid).

In a second aspect of the present invention, there is provided an early diagnosis kit for rheumatoid arthritis, the diagnosis kit comprising reagents for detecting a substance in a marker pool in sample blood, the marker pool comprising the following substances: dehydroandrosterone sulfate, glycoproline, isovironic acid, Lactic acid, caprilic acid, Chenodeoxycholic acid glycerol conjugate.

Specifically, the expression of the Dehydroepiandrosterone sulfate in OA, RAI, RAII and RAIII groups is reduced by taking the level of the marker detected in a healthy blood group as a reference; OA, RAI, RAII and RAIII groups of Lactic acid are all expressed up-regulated; the expression of the chemodeoxycholic acid glycine conjugate in OA, RAI, RAII and RAIII groups is reduced, but the expression level of the RAI group is obviously higher than that of the other three groups; the expression level of the Caprylic acid in the RAIII group is reduced compared with that in the H group, the OA group has no obvious change, and both the RAI group and the RAII group are up-regulated; only the OA group showed up-regulation of Glycylproline expression level, and the other three groups showed no significant change; the expression level of isovirac acid, OA group are up-regulated, and RAI, RAII and RAIII groups are all down-regulated.

In a third aspect of the invention, a method of screening for or identifying an agent or medicament for the treatment of early rheumatoid arthritis, the method comprising the step of detecting a change in the level of a substance in a marker pool in blood, the marker pool comprising: dehydroandrosterone sulfate, glycoproline, isovironic acid, Lactic acid, caprilic acid, Chenodeoxycholic acid glycerol conjugate.

Further, the method for screening or identifying the agent or the drug for treating early rheumatoid arthritis comprises the following steps:

(1) detecting the expression (or content) level of a marker pool in the blood of a rheumatoid arthritis test subject;

(2) administration: administering a candidate agent or drug candidate to a subject with rheumatoid arthritis;

(3) detecting the expression (or content) level of the marker pool in the blood of the rheumatoid arthritis test object after administration, and comparing the change of the expression (or content) level of the substance in the marker pool detected twice.

In a fourth aspect of the present invention, there is provided an assay for screening the above marker pools, the assay comprising the steps of:

respectively pretreating a healthy blood sample, an OA patient blood sample and an RA patient blood sample by adopting an organic solvent, separating and determining chemical components in the pretreated samples by using an ultra-high liquid chromatography-mass spectrometry method, then preprocessing UHPLC-MS raw data of the obtained healthy blood sample, the OA patient blood sample and the RA patient blood sample, and finally screening the marker pool by using a multivariate statistical analysis method and a univariate analysis method.

In order to obtain as much metabolite information of the blood sample as possible, preferably, the pretreatment process includes: adding anticoagulant into healthy blood sample, OA patient blood sample and RA patient blood sample respectively to obtain plasma, mixing with the extractive solution containing internal standard, and performing vortex, ultrasonic treatment, centrifugation and drying treatment. Then re-dissolving with a re-solution, and performing vortex, ultrasonic treatment and centrifugal treatment.

In order to separate and identify each compound in a blood metabolite sample, appropriate chromatographic and mass spectrometric analysis conditions need to be selected. The invention researches the test conditions of the ultra-high performance liquid chromatography condition aiming at the characteristics of the chemical components of the blood sample, and screens the ultra-high performance liquid chromatography condition which ensures that the separation effect of each component of the analysis sample is better. Preferably, the ultra performance liquid chromatography conditions are: the invention uses EXIONLC System (SCIEX) ultra high performance liquid chromatograph to perform chromatographic separation on target compounds through a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm, Waters) liquid chromatographic column. The liquid chromatogram is characterized in that the phase A is an aqueous solution containing 5mM ammonium acetate, and the phase B is acetonitrile. The column oven temperature was 40 ℃, the sample plate set at 4 ℃ and the sample introduction volume was 2 μ L.

The invention uses SCIEX 6500QTRAP + triple quadrupole mass spectrometer equipped with Iondrive Turbo V ESI ion source to perform mass spectrum analysis in a Multiple Reaction Monitoring (MRM) mode. The ion source parameters were as follows: curtain Gas 40psi, IonSpray Voltage 4500V, Temperature 475 deg.C, Ion Source Gas 1 30psi, Ion Source Gas 2 30 psi.

Preferably, the obtained UHPLC-MS raw data is converted from protewizard into mzXML file, and analyzed by R software package XCMS (v 3.2). Data preprocessing includes peak identification, peak alignment, peak extraction, retention time correction, peak integration, etc.

Preferably, to ensure reproducibility of metabolomic data, peaks with Relative Standard Derivatives (RSDs) of more than 30% in quality control samples are excluded. The left peak was annotated by using the R package CAMERA and compared to Retention Time (RT) and mass to charge ratio (m/z) indicators in the library. After that, we obtained data consisting of peak intensity, RT and m/z, and further removed more than 50% of missing peaks in the sample (intensity ═ 0). Multivariate and univariate analyses were performed after normalization with internal standard peak intensities.

Preferably, the multivariate statistical analysis method is an orthogonal-partial least squares projection discriminant analysis (OPLS-DA) method.

Preferably, the univariate analysis method is the Wilcoxon-Mann-Whitney test method.

Further preferably, a VIP value greater than 1 is set in the OPLS-DA assay and a P value less than 0.05 in the univariate assay.

Compared with the related technology known by the inventor, one technical scheme of the invention has the following beneficial effects:

the invention provides a marker pool of a new metabolome in blood and a corresponding diagnostic kit, the kit can be used for diagnosing early rheumatoid arthritis by detecting metabolites in blood, has high accuracy, can discover the rheumatoid arthritis as early as possible, can classify the rheumatoid arthritis, can distinguish the rheumatoid arthritis from osteoarthritis, and can intervene and treat diseases as early as possible. The invention is helpful to identify potential biological relations and improve the understanding of the whole biological mechanism through the comprehensive analysis of metabonomics data.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention.

FIG. 1 is a composition of a marker pool of the present invention.

Fig. 2A and 2B are examples of extracted ion chromatograms; ion chromatograms of the standard solution (fig. 2A) and the sample (fig. 2B) were extracted. The peaks are shown in sequence.

FIG. 3 shows the content changes of the H, OA, RAI, RAII and RAIII markers of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

Analysis method for screening blood marker pool

Subject: RA patients, OA patients and healthy volunteers.

2ml of peripheral blood of the subject is respectively extracted, and anticoagulant is added into a blood collection tube. Fresh blood samples are centrifuged and immediately processed after being layered, and if the fresh blood samples cannot be immediately processed, the fresh blood samples need to be stored at low temperature. Thawing fresh blood sample or frozen blood sample in ice water bath, vortexing for 30s, mixing 100 μ L plasma with 400 μ L extractive solution containing internal standard (methanol: acetonitrile: 1(v/v)), vortexing for 30s, and performing ice water bath ultrasound for 10 min; placing in a refrigerator at-40 deg.C for 1 h. The sample solution was then centrifuged at 12000rpm for 10min at 4 ℃. 100 μ L of the supernatant was dried in a freeze concentration centrifugal drier. Re-dissolving with 100 μ L of a heavy solution (acetonitrile: pure water 1:9(v/v)), vortexing for 30s, and performing ultrasonic treatment in ice water bath for 10min, and centrifuging the sample solution at 12000rpm at 4 deg.C for 10 min; about 100. mu.L of the supernatant was transferred to a sample bottle and subjected to the machine test.

The invention uses EXIONLC System (SCIEX) ultra high performance liquid chromatograph to perform chromatographic separation on target compounds through a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm, Waters) liquid chromatographic column. The liquid chromatogram is characterized in that the phase A is an aqueous solution containing 5mM ammonium acetate, and the phase B is acetonitrile. The column oven temperature was 40 ℃, the sample plate set at 4 ℃ and the sample introduction volume was 2 μ L.

The invention uses SCIEX 6500QTRAP + triple quadrupole mass spectrometer equipped with Iondrive Turbo V ESI ion source to perform mass spectrum analysis in a Multiple Reaction Monitoring (MRM) mode. The ion source parameters were as follows: curtain Gas 40psi, IonSpray Voltage 4500V, Temperature 475 deg.C, Ion Source Gas 1 30psi, Ion Source Gas 2 30 psi.

And (4) analyzing LC-MS metabonomics data. The raw data obtained were converted from proteo wizard to mzXML files and analyzed by the R software package XCMS (v 3.2). Data preprocessing includes peak identification, peak alignment, peak extraction, retention time correction, peak integration, etc. To ensure reproducibility of metabolomics data, peaks with Relative Standard Derivatives (RSDs) greater than 30% in quality control samples were excluded. The left peak was annotated by using the R package CAMERA and compared to Retention Time (RT) and mass to charge ratio (m/z) indicators in the library. After that, we obtained data consisting of peak intensity, RT and m/z, and further removed more than 50% of missing peaks in the sample (intensity ═ 0). Multivariate and univariate analyses were performed after normalization with internal standard peak intensities. Multivariate statistical analyses, such as PCA and OPLS-DA, were performed by SIMCA software (Umetrics, Sweden). At the same time, univariate analysis (Wilcoxon-Mann-Whitney test) was applied to determine the different metabolites. Metabolites with a VIP value greater than 1 in the OPLS-DA assay and a P value less than 0.05 in the univariate assay were identified as significantly altered metabolites, i.e. as marker pools, comprising the following: dehydroandrosterone sulfate, glycoproline, isovironic acid, Lactic acid, caprilic acid, chemodeoxycholic acid glycol conjugate, as shown in FIG. 1.

Example 2

The application method of the kit comprises the following steps: 2ml of peripheral blood of the subject is respectively extracted, and anticoagulant is added into a blood collection tube. Fresh blood samples are centrifuged and immediately processed after being layered, and if the fresh blood samples cannot be immediately processed, the fresh blood samples need to be stored at low temperature. Thawing fresh blood sample or frozen blood sample in ice water bath, vortexing for 30s, mixing 100 μ L plasma with 400 μ L extractive solution containing internal standard (methanol: acetonitrile: 1(v/v)), vortexing for 30s, and performing ice water bath ultrasound for 10 min; placing in a refrigerator at-40 deg.C for 1 h. The sample solution was then centrifuged at 12000rpm for 10min at 4 ℃. 100 μ L of the supernatant was dried in a freeze concentration centrifugal drier. Re-dissolving with 100 μ L of a heavy solution (acetonitrile: pure water 1:9(v/v)), vortexing for 30s, and performing ultrasonic treatment in ice water bath for 10min, and centrifuging the sample solution at 12000rpm at 4 deg.C for 10 min; about 100. mu.L of the supernatant was transferred to a sample bottle and subjected to the machine test.

Accurately weighing corresponding amount of standard substance in a 10mL volumetric flask, and respectively preparing into 10mmol/L standard substance stock solutions. And (3) taking a corresponding amount of standard substance stock solution into a 10mL volumetric flask to prepare a mixed standard solution. The standard solutions were diluted sequentially to obtain a series of calibration solutions, and the standard information is shown in table 1.

TABLE 1 Standard substance information

The invention uses EXIONLC System (SCIEX) ultra high performance liquid chromatograph to perform chromatographic separation on target compounds through a Waters ACQUITY UPLC HSS T3 (100X 2.1mm,1.8 μm, Waters) liquid chromatographic column. The liquid chromatogram is characterized in that the phase A is an aqueous solution containing 5mM ammonium acetate, and the phase B is acetonitrile. The column oven temperature was 40 ℃, the sample plate set at 4 ℃ and the sample introduction volume was 2 μ L.

The invention uses SCIEX 6500QTRAP + triple quadrupole mass spectrometer equipped with Iondrive Turbo V ESI ion source to perform mass spectrum analysis in a Multiple Reaction Monitoring (MRM) mode. The ion source parameters were as follows: curtain Gas 40psi, IonSpray Voltage 4500V, Temperature 475 deg.C, Ion Source Gas 1 30psi, Ion Source Gas 2 30 psi.

Standard solutions of the target compounds were introduced into the mass spectra before UHPLC-MS/MS analysis. For each target compound, its MRM parameters were optimized, and the specific parameters are shown in table 2.

TABLE 2 MRM parameters

The calibration solutions were subjected to UPLC-MRM-MS/MS analysis using the methods described previously. After calibration, the solution is diluted by 2 times in sequence and then subjected to UHPLC-MRM-MS analysis, and the detection limit and the quantification limit of the method are calculated according to the signal-to-noise ratio of the solution. The method minimum detection limit (LLOD) is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 3, and the method minimum quantitation limit (LLOQ) is defined as the concentration of the compound corresponding to a signal-to-noise ratio of 10 (US FDA gulideline for biological assay).

The precision of the method was assessed by the standard relative deviation (RSD) of QC sample replicate injections. The accuracy was assessed by the Recovery (Recovery) of the QC sample, and the percentage value of the measured concentration to the spiked concentration was the spiked Recovery.

Standard solutions and sample Extraction Ion Chromatograms (EICs) are shown in fig. 2A and 2B, from which it can be seen that: firstly, most target compounds present symmetrical chromatographic peaks by adopting an analysis method; secondly, the chromatographic separation of each target compound is well realized; and thirdly, the retention time and the chromatographic peak shape of the target compound in the biological sample and the standard solution have no obvious difference.

The quantitative parameters of the target compound are shown in Table 3, the lowest detection limit (LLODs) is between 1.22 and 625nmol/L, the lowest quantitative limit (LLOQs) is between 2.44 and 1250nmol/L, and the regression coefficient is above 0.9985.

TABLE 3 quantitative parameters of the target Compounds

The recovery (recovery) and the standard relative deviation (RSD) of the QC samples are shown in Table 4, and the number of times of repeated injection of the QC samples is 35. As shown in the table, the average recovery of all target compounds was between 95.0% and 110.3%, with a standard relative deviation of less than 13.8%.

TABLE 4 recovery (recovery) and standard relative deviation (RSD) of QC samples

The above data indicate that the method can accurately and reliably detect the content of the target metabolite in the sample within the concentration range shown above. And further analyzing to obtain the marker pool index.

Example 3

The inventors of the present invention collected 174 blood samples of RA patients (RA group) and 66 blood samples of OA patients (OA group) for assay analysis, and collected 51 healthy blood samples (H group) as a control group in the physical examination center. The kit of example 2 was used to perform the method, and the results are shown in FIG. 3 and Table 5: the levels of the markers detected in the group H are used as reference, and the expression of dehydroepisterone sulfate in the groups OA, RAI, RAII and RAIII is reduced; OA, RAI, RAII and RAIII groups of Lactic acid are all expressed up-regulated; the expression of the chemodeoxycholic acid glycine conjugate in OA, RAI, RAII and RAIII groups is reduced, but the expression level of the RAI group is obviously higher than that of the other three groups; the expression level of the Caprylic acid in the RAIII group is reduced compared with that in the H group, the OA group has no obvious change, and both the RAI group and the RAII group are up-regulated; only the OA group shows up-regulation on the expression level of the glycoproline, and the other three groups have no obvious change; the expression level of isovirac acid, OA group are up-regulated, and RAI, RAII and RAIII groups are all down-regulated. The results in Table 5 show that the blood markers are important in RA diagnosis.

TABLE 5

Note: "-" means no significant change

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. The application of the following substances as a marker pool in preparing or screening a rheumatoid arthritis diagnostic reagent or an early diagnosis kit is characterized in that the marker pool comprises the following substances: dehydroepiandrosterone glycosulfate (Dehydroepiandrosterone sulfate), glycine-L-proline (glycoproline), Isovaleric acid (Isovaleric acid), Lactic acid (Lactic acid), Caprylic acid (Caprylic acid), and glycochenodeoxycholic acid (Chenodeoxycholic acid).

2. An early diagnosis kit for rheumatoid arthritis, comprising a reagent for detecting a substance in the marker pool according to claim 1 in sample blood.

3. The diagnostic kit according to claim 2, wherein the expression of all of OA, RAI, RAII, and RAIII dehydroepisterone sulfate is down-regulated with reference to the levels of the markers detected in the healthy blood group; OA, RAI, RAII and RAIII groups of Lactic acid are all expressed up-regulated; the expression of the chemodeoxycholic acid glycine conjugate in OA, RAI, RAII and RAIII groups is reduced, and the expression level of the RAI group is obviously higher than that of the other three groups; the expression level of the Caprylic acid in the RAIII group is reduced compared with that in the H group, the OA group has no obvious change, and both the RAI group and the RAII group are up-regulated; for the expression level of glycoproline, OA group showed up-regulation, and the other three groups showed no significant change; for the expression level of isovariac acid, the OA group is up-regulated, and the RAI, RAII and RAIII groups are all down-regulated.

4. A method for screening or identifying an agent or drug for the treatment of early stage rheumatoid arthritis, the method comprising the step of detecting a change in the level of a substance in the blood in the marker pool of claim 1.

5. The method of claim 4, wherein the method of screening or identifying an agent or drug for the treatment of early stage rheumatoid arthritis comprises the steps of:

(1) detecting the level of expression or content of a marker pool in blood of a rheumatoid arthritis test subject;

(2) administration: administering a candidate agent or drug candidate to a subject with rheumatoid arthritis;

(3) detecting the expression or content level of the marker pool in the blood of the rheumatoid arthritis test object after administration, and comparing the change of the expression or content level of the substance in the marker pool detected twice.

6. An assay for screening the marker pool of claim 1, comprising the steps of:

respectively pretreating a healthy blood sample, an OA patient blood sample and an RA patient blood sample by adopting an organic solvent, separating and determining chemical components in the pretreated samples by using an ultra-high liquid chromatography-mass spectrometry method, then preprocessing UHPLC-MS raw data of the obtained healthy blood sample, the OA patient blood sample and the RA patient blood sample, and finally screening the marker pool by using a multivariate statistical analysis method and a univariate analysis method.

7. The analytical method of claim 6, wherein the preprocessing comprises: adding an anticoagulant to a healthy blood sample, an OA patient blood sample and an RA patient blood sample respectively to obtain plasma, mixing the plasma with an extracting solution containing an internal standard, and performing vortex, ultrasonic, centrifugal and drying treatment; then re-dissolving with a re-solution, and performing vortex, ultrasonic treatment and centrifugal treatment.

8. The analytical method of claim 6, wherein the ultra performance liquid chromatography conditions are: the liquid chromatogram is characterized in that the phase A is an aqueous solution containing 5mM ammonium acetate, the phase B is acetonitrile, the temperature of a column incubator is 40 ℃, a sample plate is set to be 4 ℃, and the injection volume is 2 mu L.

9. The analytical method of claim 6, wherein the mass spectrometry conditions are: mass spectrometry was performed in the Multiple Reaction Monitoring (MRM) mode using SCIEX 6500QTRAP + triple quadrupole mass spectrometer equipped with an IonDrive Turbo V ESI ion source; the ion source parameters were as follows: curtain Gas 40psi, IonSpray Voltage 4500V, Temperature 475 deg.C, Ion Source Gas 1 30psi, Ion Source Gas 2 30 psi.

10. The analytical method as defined in claim 6, wherein the obtained UHPLC-MS raw data is converted from proteo wizard into mzXML file, and analyzed by R software package XCMS (v 3.2); the data preprocessing comprises peak identification, peak alignment, peak extraction, retention time correction and peak integration;

preferably, peaks with Relative Standard Derivatives (RSDs) greater than 30% in the quality control sample are excluded; annotation of the left peak by using the R package CAMERA, compared to Retention Time (RT) and mass to charge ratio (m/z) indices in the library; thereafter, data consisting of peak intensity, RT, and m/z were obtained, and further peaks missing in 50% or more of the samples were removed (intensity ═ 0); multivariate and univariate analysis is carried out after normalization is carried out by using the internal standard peak intensity;

preferably, the multivariate statistical analysis method is an orthogonal-partial least squares projection discriminant analysis (OPLS-DA) method;

preferably, the univariate analysis method is the Wilcoxon-Mann-Whitney test method;

further preferably, a VIP value greater than 1 is set in the OPLS-DA assay and a P value less than 0.05 in the univariate assay.

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